Volume 67, Issue 5, Pages 733-743.e4 (September 2017) Structural and Functional Impacts of ER Coactivator Sequential Recruitment  Ping Yi, Zhao Wang, Qin Feng, Chao-Kai Chou, Grigore D. Pintilie, Hong Shen, Charles E. Foulds, Guizhen Fan, Irina Serysheva, Steven J. Ludtke, Michael F. Schmid, Mien-Chie Hung, Wah Chiu, Bert W. O’Malley  Molecular Cell  Volume 67, Issue 5, Pages 733-743.e4 (September 2017) DOI: 10.1016/j.molcel.2017.07.026 Copyright © 2017 Elsevier Inc. Terms and Conditions

Molecular Cell 2017 67, 733-743.e4DOI: (10.1016/j.molcel.2017.07.026) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 ER Sequentially Recruits Different Coactivators (A) CARM1 recruitment to the ER complex followed behind the recruitment of SRC-3. Shown are the ChIP results of ERα, SRC-3, and CARM1 in MCF-7 cells stimulated with 10 nM E2 at different time points at the ER target gene TFF1 promoter. (B) CARM1 synergized with SRC-3 and p300 to activate ER-mediated transcription. The ER-mediated transcription activity was measured through an ERE-driven luciferase reporter in the absence or presence of 10 nM 17-β estradiol (E2). Data are represented as mean ± SEM. Molecular Cell 2017 67, 733-743.e4DOI: (10.1016/j.molcel.2017.07.026) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 Classification of ERE/ER/CoA Complexes (A and B) Workflow for stepwise 3D classification of ERα + SRC-3 + p300 + CARM1 (A) and of ERα + SRC-3 + p300 + CARM1 + Fab (B) datasets using new multirefine and e2refinemulti in EMAN2. Each subset was refined separately with the specified particle number. Each map was segmented to annotate different proteins: p300, blue; ER, green; SRC-3a/b, orange and red, respectively; CARM1, pink; CARM1 Fab, yellow. Molecular Cell 2017 67, 733-743.e4DOI: (10.1016/j.molcel.2017.07.026) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 CARM1 Overexpression Reduced the SRC-3:ER Ratio, and Its N-Terminal Domain Mediated the Interaction with p300 (A) Raw flow-proteometric analysis data for a single ER/SRC-3 complex in HeLa cells without (left panel) or with (right panel) CARM1 overexpression. (B) Box whisker plot of SRC-3 and ERα fluorescence intensities detected in single ER/SRC-3 complexes without or with CARM1 expression. (C) The N-terminal domain of CARM1 (1–140 aa) mediated the interaction between CARM1 and p300 in vitro. Shown is the GST pull-down experiment using GST-fused CARM1 full length and different fragments to pull-down purified p300 protein. (D) CARM1 N-terminal domain deletion significantly reduced its ability to interact with p300 and ER in cells. Shown is a coIP experiment. Molecular Cell 2017 67, 733-743.e4DOI: (10.1016/j.molcel.2017.07.026) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 Communication between p300 and CARM1 within the ER Complex Regulates HAT and HMT Activity (A) Segmented p300 from map 1A. (B) Segmented p300 from map 1C. (C) CARM1 increased p300 autoacetylation and its HAT activity on histone H3 in vitro. Shown is the autoradiograph of 3H-labeled acetylation. (D) Coomassie blue staining of purified GST-CARM1 WT and Δ1-140 mutant from bacteria. (E) Deletion of the N-terminal domain abolished the ability of CARM1 to regulate p300 HAT activity in vitro. Top panel: the autoradiograph of 3H-labeled acetylation. Bottom panels: western blot analysis on the levels of ER, SRC-3, p300, and CARM1 WT or Δ1-140 mutant in the input. ∗, the uncleaved GST-CARM1; #, GST-removed CARM1 through thrombin cleavage. (F) CARM1 selectively increased p300 HAT activity on H3K18. Shown is the western blot analysis of the levels of histone acetylation in the in vitro HAT assay using different acetylation antibodies. (G) p300-mediated acetylation significantly increased CARM1-mediated H3R17 methylation. Shown is the autoradiograph of 3H-labeled methylation in vitro. Molecular Cell 2017 67, 733-743.e4DOI: (10.1016/j.molcel.2017.07.026) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 5 A Working Model of the Impact of Coactivator Sequential Binding on ER-Mediated Transcriptional Activation Upon estrogen stimulation, an ER dimer binds two SRC-3 molecules which then recruit p300. The binding of p300 promotes the basal level of histone H3K18 acetylation at the ER binding sites. The recruitment of CARM1 displaces one SRC-3 molecule from the complex and induces a p300 conformational change to enhance p300-mediated H3K18 acetylation. Increased H3K18ac further promotes CARM1-mediated H3R18 dimethylation. These histone epigenetic modifications then bring in reader proteins to promote transcription. Molecular Cell 2017 67, 733-743.e4DOI: (10.1016/j.molcel.2017.07.026) Copyright © 2017 Elsevier Inc. Terms and Conditions